ES2618402T3 - Modified peptides and their use to treat autoimmune diseases - Google Patents

Abstract

A peptide, or salt thereof, which comprises or consists of the amino acid sequence of SEQ ID NO: 2 IHMVYSKRSGKPRGYAFIEY [SEQ ID NO: 2], which comprises a phosphoserine at position 9 and an oxidized methionine at position 3.

Description

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

DESCRIPTION

Modified peptides and their use to treat autoimmune diseases Field of the invention

The present invention relates to a modified peptide and its use to treat autoimmune diseases. Background

Autoimmune diseases are caused by an overactive immune response of the immune system against substances and tissues normally present in a multicellular organism, that is, the immune system attacks its own components. This can be restricted to certain organs or involve a particular tissue in different places. The treatment of autoimmune diseases is typically performed with an immunosuppressive medication that reduces the entire immune response.

Lupus is an autoimmune disease that is estimated to affect almost 1.4 million Americans, mainly women between the ages of 20 and 40. Antibodies that attack connective tissue are involved in lupus. The main form of lupus is a systematic form (systemic lupus erythematosus, SLE). SLE is a chronic autoimmune disease with strong genetic as well as environmental components (see, for example, Hochberg MC, Dubois' Lupus Erythematosus. 5th ed., Wallace DJ, Hahn BH, eds. Baltimore: Williams and Wilkins (1997); Wakeland EK, et al., Immunity 2001; 15 (3): 397-408; Nath SK, et al., Curr. Opin. Immunol. 2004; 16 (6): 794-800; D'Cruz et al., Lancet (2007), 369: 587-596). Various additional forms of lupus are known including, but not limited to, cutaneous lupus erythematosus (LEC), lupus nephropathy (NL), neuropsychiatric lupus (LESNP) and neonatal lupus.

Lupus without treatment can be fatal, as the attack progresses from the skin and joints to the internal organs, including the lungs, heart and kidneys (kidney disease being the main concern), which makes early diagnosis and accurate and / or the risk assessment of developing lupus is particularly critical. Lupus appears mainly as a series of outbreaks, with intermediate periods with little or no manifestation of the disease. Renal injury, measured by the amount of protein in the urine (proteinuria) is one of the most precise areas of lesions associated with pathogenicity in SLE, and is responsible for at least 50% of the disease mortality and morbidity , without any treatment.

Clinically, SLE is a heterogeneous disorder characterized by high affinity autoantibodies (auto-Ac). AutoAbs play an important role in the pathogenesis of SLE, and the various clinical manifestations of the disease are due to the deposition of immune complexes that contain antibodies in blood vessels that leads to inflammation in the kidney, brain and skin. Auto-Ac also have direct pathogenic effects that contribute to hemolytic anemia and thrombocytopenia. SLE is associated with the production of antinuclear antibodies, circulating immune complexes and complement system defects. SLE has an incidence of approximately 1 in 700 women between the ages of 20 and 60 in the black population. SLE can affect any organ system and can cause serious tissue damage. In the SLE numerous autoAb of different specificity are present. Patients with SLE frequently produce auto-Ac that have specific anti-DNA, anti-Ro and antiplatelet specificities and are capable of initiating clinical features of the disease, such as glomerulonephritis, arthritis, serositis, complete heart block in newborns and abnormalities. Hematological These autoAb are also possibly related to alterations of the central nervous system. Arbuckle et al. described the development of auto-Ac before the clinical onset of SLE (Arbuckle et al. N. Engl. J. Med. 349 (16): 1526-1533 (2003)).

AutoAc that recognize RNA binding proteins (RBP; also called extractable nuclear antigens) were first characterized in SLE over 40 years ago (Holman, Ann NY Acad. Sci. 124 (2): 800-6 (1965) ). Said RBP comprises a group of proteins-SSA (Ro52 / TRIM21 and Ro60 / TROVE2), SSB (La), small nuclear ribonucleoprotein (RNP) U1 and Smith protein (Sm) with roles in RNA processing and biochemistry. Auto-Ac IgG anti-SSA and anti-SSB have been found not only in SLE, but also in Sjogren's syndrome and rheumatoid arthritis. Auto-Ac anti-SSA are associated with subacute cutaneous lupus erythematosus, and with congenital heart block and neonatal lupus in children born to women positive for anti-SSA. Anti-SSB auto-Acs are almost always found together with anti-SSA auto-Ac, and both autoantigens are associated with cytoplasmic hYRNA (Lerner et al., Science 211 (4480): 400-2 (1981)). Auto-Ac anti-Sm are very specific for SLE and are usually found together with auto-Ac anti-RNP. Both Sm and RNP proteins are associated with RNAs species in the splenic nuclear RNA (Lerner et al., Proc Natl Acad Sci USA 76 (11): 5495-9 (1979)). Auto-Ac anti-RNP is also found in patients with mixed connective tissue disease. It has been suggested that the presence of auto-Ac anti-RNP can identify cases of SLE that show less lasting responses after a B-lymphocyte reduction therapy (Cambridge et al., Ann Rheum Dis 67: 1011-16 (2008)) .

The prior art discloses medicines for the treatment of autoimmune diseases. For example, the two international applications WO 03/020747 and WO 03/025014 disclose a 70 kDa snRNP fragment, which is modified by phosphorylation and / or acetylation, to treat said pathologies, i.e. diseases

5

10

fifteen

twenty

25

30

35

Autoimmune However, additional experimentation has indicated that the peptides disclosed in WO 03/020747 and WO 03/025014 are relatively unstable and are rapidly removed when administered to mammals. In addition, some of the unveiled peptides are inactive.

Therefore, there is a need to provide a better treatment for autoimmune pathologies.

Summary

The present description provides compositions and their use in the treatment of an autoimmune disease that is based on the surprising and unexpected discovery that certain modified forms of the peptide of SEQ ID NO: 2 are more stable when administered to mammals. Thus, in one aspect, the present description provides compositions comprising these forms and a pharmaceutically acceptable carrier.

In another aspect, the present description provides a pharmaceutical composition, or drug, useful for improving autoimmune diseases, advantageously for the treatment of lupus.

In a further aspect, the present description provides the pharmaceutical composition for use in the treatment of an autoimmune disease comprising administering an effective amount of a therapeutic composition as described herein.

In certain embodiments, the description provides an isolated peptide (recombinant or synthesized), or a salt thereof, which comprises or consists of the amino acid sequence:

IHMVYSKRSGKPRGYAFIEY [SEQ ID NO: 2],

wherein the serine (S) in position 9 is phosphorylated, and the methionine (M) in position 3 is oxidized.

In certain embodiments, the description provides a peptide of compound I having the following formula:

image 1

Compound I can also be represented by:

HMI (O) VYSKRS (PO3H2) GKPRGYAFIEY [SEQ ID NO: 5]

Where "M (O)" represents oxidized methionine, and "S (PO3H2)" represents phosphoserine.

These peptides come from the human protein U1 snRNP 70 kDa (SEQ ID NO: 3) and correspond to the region delimited by the amino acid segment that extends from the remainder 132 to the remainder 151 of SEQ ID NO: 3. Formally, the The remainder that is phosphorylated corresponds to the amino acid at position 140 from the first methionine of SEQ ID NO: 3 and the remainder that is oxidized corresponds to the amino acid at position 134 from the first methionine of SEQ ID NO: 3.

In certain aspects, the description provides an isolated peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or salt thereof, which comprises a phosphoserine at position 10, and an oxidized methionine residue at position 4 In one embodiment of this aspect, the description provides a composition comprising an isolated peptide having or consisting of the amino acid sequence of SEQ ID NO: 1, or salt thereof, in which the peptide comprises a phosphoserine in the position 10 and an oxidized methionine residue at position 4. In certain additional embodiments, the peptide of SEQ ID NO: 1 also comprises an acetylated lysine residue. In particular, said peptide of SEQ ID NO: 1 comprises a phosphoserine in position 10 and an oxidized methionine residue in position 4, and an acetylation of one or both lysines in position 8 and 12 and, more particularly, also comprises a phosphoserine in position 7.

In one embodiment of this aspect, the description provides a composition comprising an isolated peptide having or consisting of the amino acid sequence of SEQ ID NO: 2, or salt thereof, wherein the peptide comprises a phosphoserine at position 9 and an oxidized methionine residue at position 3. In certain additional embodiments, the peptide of SEQ ID NO: 2 also comprises an acetylated lysine residue.

In certain aspects, the description provides a composition comprising an effective amount of one or more of the peptides described herein. In certain embodiments, the composition further comprises a

5

10

fifteen

twenty

25

30

35

40

pharmaceutically acceptable vehicle. In a further embodiment, the description provides a composition comprising an effective amount of at least one peptide, or salt thereof, selected from the group consisting of the amino acid sequence SEQ ID NO: 2, which comprises a phosphoserine at position 9 , and oxidized methionine at position 3; amino acid sequence SEQ ID NO: 1, which comprises a phosphoserine at position 10 and an oxidized methionine at position 4; and a combination of both.

In certain embodiments, the description provides a peptide of compound II having the following formula:

image2

Compound II can also be represented by:

RIHM (O) VYSKRS (PO3H2) GKPRGYAFIEY [SEQ ID NO: 4]

in which M (O) represents the oxidation of methionine and S (PO3H2) represents phosphorylation of serine.

Thus, the description provides peptides, or a salt thereof, which comprise or consist of the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

The invention will be better illustrated by the following figures 1 to 6, the accompanying description, the examples and the appended claims.

Brief description of the drawings

An embodiment of the invention should not be considered as limiting the invention.

Figure 1 represents the stability at 37 ° C of the peptide according to the invention (Compound II) in comparison with the stability of the peptide consisting of SEQ ID NO: 1, in which the serine in position 10 is phosphorylated. The graph represents the percentage of stability over time (expressed in days). Curves A-C represent the stability of Compound II at a concentration of 200, 100 and 50 pg / ml, respectively. The D-F curves represent the stability of the peptide consisting of SEQ ID NO: 1, in which the serine in position 10 is phosphorylated at a concentration of 200, 100 and 50 pg / ml, respectively.

Figure 2 is a Kaplan-Meier plot representing the cumulative survival rate (in percentage) over time (expressed in weeks) of mice in which NaCl (line with circles) has been injected, the peptide consisting of SEQ ID NO: 1, in which the serine in position 10 is phosphorylated (line with squares) and compound II according to the invention (lines with triangles).

Figure 3 represents the proteinuria score over time (expressed in weeks) of mice in which NaCl (line with circles) has been injected, the peptide consisting of SEQ ID NO: 1, in which the serine is in position 10 is phosphorylated (line with squares) and compound II according to the invention (lines with triangles).

Figure 4 represents the measure of the cell hypercellularity of MRL / lpr mice. The Y axis represents the number of cells / ml of blood (x 106), in mice treated with NaCl (circles), the peptide consisting of SEQ ID NO: 1, in which the serine at position 10 is phosphorylated (squares) ) and compound II according to the invention (triangles).

Figure 5 represents the measure of affinity for peptide HSC70 protein consisting of SEQ ID NO: 1, in which the serine at position 10 is phosphorylated. The curves correspond to the Biacore response over time (expressed in seconds) using the peptide consisting of SEQ ID NO: 1, in which the serine in position 10 is phosphorylated at a concentration of 25 pM (A ), 12.5 pM (B), 6.25 pM (C), 3.12 pM (D) and 1.56 pM (E).

Figure 6 represents the measure of the affinity of compound II according to the invention for the HSC70 protein. The curves correspond to the Biacore response over time (expressed in seconds) using compound II at a concentration of 25 pM (A), 12.5 pM (B), 6.25 pM (C), 3, 12 pM (D) and 1.56 pM (E).

Detailed description

Next, a detailed description of the invention is provided to help those skilled in the art to implement the present invention. Those of ordinary skill in the art can make modifications and

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

variations in the embodiments described herein without departing from the spirit or scope of the present invention. Although any method and material similar or equivalent to those described herein may also be used in the implementation or testing of the present invention, the preferred methods and materials are now described. Unless defined otherwise, all the technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which the present invention belongs. The terminology used in the description of the invention of this document serves only to describe particular embodiments and is not intended to be limiting of the invention.

As used herein, the following terms may have the meanings attributed to them below, unless otherwise specified. However, it should be understood that other meanings known or understood by those of ordinary skill in the art are also possible, and are within the scope of the present invention.

When a range of values is provided, it is understood that all intermediate values are included within the invention, up to the tenth part of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that interval and any other indicated or intermediate value in that designated interval. Also included within the invention are the upper and lower limits of these smaller intervals that can be included independently in the smaller intervals, subject to any limit specifically excluded in the indicated range. When the indicated interval includes one or both limits, the intervals that exclude both of those included limits are also included in the invention.

It should be noted that, as used herein and in the appended claims, the singular forms "a", "a", "and", and "the", "the" include plural references (ie, refer to one, more than one or at least one) of the grammatical object of the article. As an example, "an element" means an element or more than one element.

The term "approximately", as used herein, when associated with numerical values or ranges, reflects the fact that there is a certain level of variation that is recognized and tolerated in the technique due to practical and / or theoretical limitations. . For example, a small variation due to intrinsic variances is tolerated in the way in which certain devices work and / or in the way in which measurements are taken. According to the above, the phrase "approximately" is normally used to include values within the standard deviation or typical error.

As used herein, "derivatives" are compositions formed from the native compounds directly, by modification or by partial substitution. As used herein, "analogs" are compositions that have a similar, but not identical, structure to the native compound.

The terms "effective amount / dose", "pharmaceutically effective amount / dose", "pharmaceutically effective amount / dose" or "therapeutically effective amount / dose" may refer, but not limited in any way, to that amount / dose of the Active pharmaceutical ingredient is enough to prevent, inhibit the appearance, improve, delay or treat (relieve a symptom to some extent, preferably all) the symptoms of a condition, disorder or pathology. The effective amount depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication and other factors recognized by experts in medical techniques. . In general, an amount between 0.1 mg / kg and 1000 mg / kg of body weight / day of active ingredients is administered depending on the potency of the agent. The toxicity and therapeutic efficacy of said compounds can be determined by conventional pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in the 50% of the population). The dose relationship between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / DE50. Compounds that have large therapeutic indices are preferred. Although compounds that have toxic side effects can be used, care must be taken to design a release system that directs these compounds to the site of the affected tissue to minimize possible lesions in uninfected cells and, thus, reduce side effects. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of said compounds is preferably within a range of circulating concentrations that include the ED50 with a small or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a range of circulating plasma concentrations that include IC50 (i.e., the concentration of the test compound that achieves a semi-maximum inhibition of symptoms) as determined in a cell culture. This information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

The expression "pharmaceutical composition", "therapeutic composition", "therapeutic formulation" or "pharmaceutically acceptable formulation" may mean, but in no way be limited to, a composition or formulation

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

which allows the effective distribution of an agent provided by the invention, which is in a form suitable for administration in the most suitable physical location for its desired activity, for example, systemic administration.

The term "pharmaceutically acceptable" or "pharmacologically acceptable" may mean, but in no way limited to entities and compositions that do not produce an adverse, allergic or other unwanted reaction when administered to an animal or a human being, as appropriate.

The expression "pharmaceutically acceptable vehicle" or "pharmacologically acceptable vehicle" may mean, but not limited to, each and every solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delay agents, and the like. , compatible with the pharmaceutical administration. Suitable vehicles are described in the most recent edition of Remington's Pharmaceutical Sciences, a conventional reference text in that field. Preferred examples of such vehicles or diluents include, but are not limited to, water, saline solution, finger solutions, 5% human serum dextrose and albumin solution. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media or agents for pharmaceutically active substances is well known in the art. Except when any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Complementary active compound compositions can also be incorporated into the compositions.

The term "systemic administration" refers to a route of administration that is, for example, enteric or parenteral, and results in the systematic distribution of an agent that leads to the systematic absorption or accumulation of drugs in the bloodstream, followed by its distribution throughout the entire body. Suitable forms, in part, depend on the use or the route of entry, for example, the oral, transdermal or injection route. Such forms should not prevent the composition or formulation from reaching a target cell (ie, a cell in which it is desired to release the negatively charged polymer). For example, the pharmacological compositions injected into the bloodstream must be soluble. Other factors are known in the art and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect. The routes of administration leading to systemic absorption include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. It has been shown that the rate of entry of a drug into the circulation is a function of molecular weight or size. The use of a liposome or other drug vehicle comprising the compounds of the present invention can potentially locate the drug, for example, in certain types of tissues, such as reticuloendothelial system (RES) tissues. A liposome formulation that can facilitate the association of a drug with the surface of cells such as lymphocytes and macrophages is also useful.

The term "local administration" refers to a route of administration in which the agent is released at a site that is appropriate or is nearby, for example, is at a distance less than or equal to approximately 10 cm from the site of the injury or disease.

The term "derivatives" may mean, but in no way limited to, chemical compositions, for example, nucleic acids, nucleotides, polypeptides or amino acids, formed from the native compounds directly, by modification or by partial substitution. The term "analogs" may refer to, but in no way is limited to, chemical compositions, for example, nucleic acids, nucleotides, polypeptides or amino acids that have a structure similar to, but not identical to, the native compound.

The term "conservative mutations" refers to the substitution, deletion or addition of nucleic acids that alter, add or eliminate a single amino acid or a small number of amino acids in a coding sequence in which nucleic acid alterations result in substitution. of a chemically similar amino acid. Amino acids that can serve as conservative substitutions among themselves include the following: Basic: Arginine (R), Lysine (K), Histidine (H); Acids: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); Hydrophilic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Hydrophobes: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C). In addition, sequences that differ by conservative variations are generally homologous.

By "homology" is meant the nucleotide sequence of two or more nucleic acid molecules or two or more nucleic acid or amino acid sequences that are partially or completely identical. In certain embodiments, the homologous amino acid or nucleic acid sequence has 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% sequence similarity or identity with a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1, respectively.

"Homologues" may be natural or may be created by artificial synthesis of one or more nucleic acids having related sequences, or by modification of one or more nucleic acids to produce related nucleic acids. Nucleic acids are homologous when they come, naturally or artificially, from a common ancestor sequence (for example, orthologs or paralogs). If the homology between two nucleic acids is not expressly described, the homology can be deduced by a comparison of nucleic acids between two or more sequences. If the sequences demonstrate some degree of sequence similarity, for example, greater than

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

Approximately 30% at the level of the primary amino acid structure, it is concluded that they share a common ancestor. For the purposes of the present invention, the genes are homologous if the nucleic acid sequences are sufficiently similar to allow recombination and / or hybridization under conditions of low stringency. In addition, polypeptides are considered homologous if their nucleic acid sequences are sufficiently similar to allow recombination or hybridization under conditions of low stringency, and optionally demonstrate membrane repair activity, and optionally can be recognized by (i.e. presenting a reaction crossed with) an antibody specific for an epitope contained within the amino acid sequence of at least one of SEQ ID NO: 1-6.

The term "cell" may refer to, but in no way be limited to, its usual biological sense, and does not refer to an entire multicellular organism. The cell, for example, can be in vivo, in vitro or ex vivo, for example, in cell culture, or it can be present in a multicellular organism including, for example, birds, plants and mammals such as humans, cows, sheep , apes, monkeys, pigs, dogs and cats. The cell can be prokaryotic (for example, a bacterial cell) or eukaryotic (for example, a mammalian or plant cell).

The term "host cell" may mean, but is in no way limited to, a cell that could be used to carry a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. A host cell may contain genes that are not within the native (non-recombinant) form of the cell, genes that are in the native form of the cell where the genes have been modified and reintroduced into the cell by artificial means , or an endogenous nucleic acid for the cell that has been artificially modified without removing the nucleic acid from the cell. A host cell can be eukaryotic or prokaryotic. The general growth conditions necessary for the culture of bacteria can be found in texts such as BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, vol. 1, N. R. Krieg, ed., Williams and Wilkins, Baltimore / London (1984). A "host cell" may also be one in which the endogenous or promoter genes, or both, have been modified to produce one or more of the polypeptide components of the complex of the invention.

As used herein, "P140" refers to a peptide consisting of the amino acid sequence SEQ ID NO: 1, in which the serine at position 10 is phosphorylated.

The expression "therapeutically effective amount or dose" includes a dose of a drug that is capable of achieving a therapeutic effect on a subject in need. For example, a therapeutically effective amount of a drug may be the amount that is capable of preventing or alleviating one or more symptoms associated with a disease or disorder, for example, a tissue injury or a muscle-related disease or disorder. The exact amount can be ascertained by a person skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vol. 1-3, 1992), Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999) and Remington: The Science and Practice of Pharmacy, 20s Edition, 2003, Gennaro, Ed., Lippincott, Williams and Wilkins).

A kit is any article manufactured (for example, a container or container) comprising at least one reagent, for example, a probe, to specifically detect a marker of the invention. The manufactured article can be promoted, distributed or sold as a unit to implement the methods of the present invention. Reagents included in said kit comprise probes / primers and / or antibodies for use in detecting the expression of sensitivity and resistance genes. In addition, the kits of the present invention may preferably contain instructions describing a suitable detection assay. Such kits can be conveniently used, for example, in clinical situations, for the diagnosis of patients presenting with symptoms of cancer, in particular patients presenting the possible presence of a tumor.

Unless defined otherwise, all technical and scientific terms used in this document have the same meaning commonly understood by a person skilled in the art to which the present invention belongs.

The following references provide an expert with a general definition of many of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, the Harper Collins Dictionary of Biology (1991).

In certain embodiments, the description provides an isolated peptide (recombinant or synthesized), or a salt thereof, which comprises or consists of the amino acid sequence:

IHMVYSKRSGKPRGYAFIEY [SEQ ID NO: 2],

in which the serine (S) in position 9 is phosphorylated, and the methionine (M) in position 3 is oxidized.

In certain embodiments, the description provides a peptide of compound I having the following formula:

5

10

fifteen

twenty

25

30

35

image3

HMI (O) VYSKRS (PO3H2) GKPRGYAFIEY [SEQ ID NO: 5] in which "M (O)" represents oxidized methionine and "S (PO3H2)" represents phosphoserine.

These peptides come from the human protein U1 snRNP 70 kDa (SEQ ID NO: 3) and correspond to the region delimited by the amino acid segment that extends from the remainder 132 to the remainder 151 of SEQ ID NO: 3. Formally, the The remainder that is phosphorylated corresponds to the amino acid at position 140 from the first methionine of SEQ ID NO: 3 and the remainder that is oxidized corresponds to the amino acid at position 134 from the first methionine of SEQ ID NO: 3.

In certain aspects, the description provides an isolated peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or a salt thereof, which comprises a phosphoserine at position 10 and an oxidized methionine residue at position 4 In one embodiment of this aspect, the description provides a composition comprising an isolated peptide having or consisting of the amino acid sequence of SEQ ID NO: 1, or a salt thereof, wherein the peptide comprises a phosphoserine at position 10 and an oxidized methionine residue at position 4. In certain additional embodiments, the peptide of SEQ ID NO: 1 also comprises an acetylated lysine residue.

In further aspects, the description provides an isolated peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or salt thereof, which comprises a phosphoserine at position 9 and an oxidized methionine residue at position 3. In one embodiment of this aspect, the description provides a composition comprising an isolated peptide having or consisting of the amino acid sequence of SEQ ID NO: 2, or salt thereof, where the peptide comprises a phosphoserine at position 9 and a oxidized methionine residue at position 3. In certain additional embodiments, the peptide of SEQ ID NO: 2 also comprises an acetylated lysine residue.

In certain aspects, the description provides a composition comprising an effective amount of one or more of the peptides described herein. In certain embodiments, the composition further comprises a pharmaceutically acceptable vehicle. In a further embodiment, the description provides a composition comprising an effective amount of at least one peptide, or salt thereof, selected from the group consisting of the amino acid sequence SEQ ID NO: 2, which comprises a phosphoserine at position 9 and oxidized methionine at position 3; amino acid sequence SEQ ID NO: 1, which comprises a phosphoserine at position 10 and an oxidized methionine at position 4; and a combination of both.

In certain embodiments, the description provides a peptide of compound II having the following formula:

image4

Compound II can also be represented by:

RIHM (O) VYSKRS (POsH2) GKPRGYAFIEY [SEQ ID NO: 4] in which M (O) represents oxidation and S (PO3H2) represents phosphorylation.

The description provides peptides and / or salts thereof that comprise or consist of the amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and combinations thereof, as well as compositions that understand

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

In certain embodiments, the description provides an isolated peptide having the amino acid sequence of SEQ ID NO: 1, comprising a phosphoserine at position 10 and an oxidized methionine at position 4 (eg, SEQ ID NO: 4) ( also referred to herein as Compound II or P140 (MO)).

According to the present description, the isolated peptides having the amino acid sequence of SEQ ID NO: 4 or 5, respectively, are modified by at least two post-translational modifications (modifications that occur after the synthesis of the peptides). In certain embodiments, the post-translational modification is selected from the group consisting of phosphorylation (addition of a PO3H2 phosphate), for example, phosphorylation of a serine moiety; oxidation, for example, oxidation of a methionine residue; acetylation, for example, acetylation of a lysine residue; and combinations thereof.

In a preferred embodiment, the description provides an isolated peptide having the amino acid sequence indicated in SEQ ID NO: 2, which comprises a phosphoserine at position 9 and an oxidized methionine at position 3, or a salt thereof. In certain embodiments, the description provides the peptide having the amino acid sequence indicated in SEQ ID NO: 2, which comprises a phosphoserine at position 9 and an oxidized methionine at position 3, or a salt thereof, and a vehicle, for example, a pharmaceutically acceptable vehicle. In certain additional embodiments, the description provides a composition, for example, a therapeutic composition, which comprises an effective amount of a peptide having the amino acid sequence indicated in SEQ ID NO: 2, which comprises a phosphoserine at position 9 and an oxidized methionine at position 3, or a salt thereof, and a vehicle, for example, a pharmaceutically acceptable vehicle.

In another embodiment, the description provides an isolated peptide having the amino acid sequence indicated in SEQ ID NO: 4 comprising a phosphoserine at position 10 and an oxidized methionine at position 4, or a salt thereof. In certain embodiments, the description provides the peptide having the amino acid sequence indicated in SEQ ID NO: 4, which comprises a phosphoserine at position 10 and an oxidized methionine at position 4, or a salt thereof, and a vehicle, for example, a pharmaceutically acceptable vehicle. In certain additional embodiments, the description provides a composition, for example, a therapeutic composition, comprising an effective amount of a peptide having the amino acid sequence indicated in SEQ ID NO: 4 comprising a phosphoserine at position 10, or a salt thereof, and an oxidized methionine at position 4, and a vehicle, for example, a pharmaceutically acceptable vehicle.

In another embodiment, the description provides an isolated peptide having the amino acid sequence indicated in SEQ ID NO: 5 comprising a phosphoserine at position 9 and an oxidized methionine at position 3, or a salt thereof. In certain embodiments, the description provides the peptide having the amino acid sequence indicated in SEQ ID NO: 5 comprising a phosphoserine at position 9 and an oxidized methionine at position 3, or salt thereof, and a vehicle, for example, a pharmaceutically acceptable vehicle. In certain additional embodiments, the description provides a composition, for example, a therapeutic composition, which comprises an effective amount of a peptide having the amino acid sequence indicated in SEQ ID NO: 5, which comprises a phosphoserine at position 9 and an oxidized methionine at position 3, or a salt thereof, and a vehicle, for example, a pharmaceutically acceptable vehicle.

Surprisingly and unexpectedly, it was discovered that the peptides described herein are more stable in vitro compared to their non-oxidized counterparts. Stability is measured as disclosed in the examples section. The phosphorylated-oxidized peptide degrades less spontaneously in solution than its non-oxidized counterpart, said stability increasing its biological properties.

In addition, the inventors have surprisingly identified that the oxidation of methionine increases the stability of the peptide without affecting the biological effect of said peptide, which is contrary to the teachings of the prior art. In fact, it has been abundantly reported in the art that proteins or peptides containing oxidized methionine have breaks in their three-dimensional structure and / or bioactivity. The modified peptides described herein have an affinity for the HSC70 protein essentially identical to the non-oxidized homologue as disclosed in the examples section.

In certain embodiments, oxidation occurs in Methionine (M) at position 9 of SEQ ID NO: 2, or at position 10 of SEQ ID NO: 1, which are positions equivalent to position 134 of SEQ ID NO: 3. The sulfur atom is oxidized as illustrated below.

image5

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

The above peptides (SEQ ID NO: 1, 2, 4 and 5) can be synthesized by techniques commonly used in this field, such as biological synthesis or chemical synthesis. Biological synthesis refers to the production, in vivo, in vitro or ex vivo, of the peptide of interest, by transcription and translation of a nucleic acid molecule encoding said peptides.

For example, the nucleic acid sequence:

MGNATHCAYATGGTNTAYWSNAARMGNWSNGGNAARCCNMGNGG NTAYGCNTTYATHGARTAYTRR [SEQ ID NO: 6]

it is transcribed and translated into an in vitro system or a host organism, to produce the peptide of SEQ ID NO: 1. The peptide produced in this way is purified according to well known techniques.

The chemical synthesis consists in polymerizing the desired peptide by adding the necessary amino acids. In the examples section a method is disclosed.

It is possible to chemically synthesize the peptides of SEQ ID NO: 1 and 2 by classical solid phase chemistry Fmoc (N- [9-fluorenyl] methoxycarbonyl) and purify them by reverse phase high performance liquid chromatography (HPLC; Neimark and Briand , 1993. Monneaux et al., 2003, Eur. J. Immunol., 33, 287-296, Page et al., 2009, PloS ONE 4, e5273).

It is also possible to directly synthesize the peptides of SEQ ID NO: 1 and 2, in which the respective residues in positions 9 and 10 are phosphorylated. For this purpose, during the peptide synthesis a serine derivative of the Fmoc-Ser (PO (Obz) OH) -OH type was used in the desired position.

A phosphate group (-PO3H2) may also be added after peptide synthesis, in accordance with protocols well known in the art.

The serine can be phosphorylated by incubation of the peptides of SEQ ID NO: 1 or 2 with specific serine kinases selected from Protema Kinase A or C (PKA or PKC) or casema kinase II, in the presence of adenosine triphosphate (ATP). In this way the peptides are phosphorylated in a serine (in position 6 or 9 of SEQ ID NO: 2, or in position 7 or 10 of SEQ ID NO: 1) or in both serines. The desired phosphorylated peptide is separated from the others, for example, by chromatography.

Chemically -PO3H2 can also be added in the specific position (in position 9 of SEQ ID NO: 2, or in position 10 of SEQ ID NO: 1) using a specific protective group, which the expert can easily choose according to With your common knowledge.

Any other technique known in this field can be used, which allows specific serine phosphorylation.

In certain embodiments, methionine oxidation is performed according to the following process: treatment with 20 mM H2O2 at 37 ° C for 4 hours or

in a solution of dimethylsulfoxide (DMSO, Me2SO), 0.1 M plus 0.5 M HCl, at 22 ° C for a period of 30 to 180 min.

Any other technique known in this field can be used, which allows the specific oxidation of methionine.

In any of the aspects or embodiments described herein, the peptide or peptides provided by the description may be present in the form of a salt known to a person skilled in the art, such as, for example, sodium salts, ammonium salts. Calcium salts, magnesium salts, potassium salts, acetate salts, carbonate salts, citrate salts, chloride salts, sulfate salts, amino hydrochloride salts, borhydrate salts, benzenesulfonate salts, phosphate salts, dihydrogen phosphate salts, succinate salts, citrate salts, salts tartrate, lactate salts, mandelate salts, methanesulfonate salts (mesylate) or p-toluenesulfonate salts (tosylate). This list is provided by way of example and is not intended to be limiting of the present invention. For example, the person skilled in the art can easily determine, according to his knowledge, the appropriate salt.

In a further embodiment, the description provides a peptide that comprises or consists of the amino acid sequence:

RIHMVYSKRSGKPRGYAFIEY [SEQ ID NO: 1],

which comprises a phosphoserine in position 10, and an oxidized methionine in position 4, or salt thereof. In an advantageous embodiment, the invention relates to the peptide defined above, which consists of the amino acid sequence SEQ ID NO: 4 or a salt thereof.

In another aspect, the description provides a therapeutic composition comprising at least one peptide as described herein, or a salt thereof, and a drug having an immunosuppressive effect. In certain embodiments, the therapeutic composition comprises an effective amount of a peptide as described herein, and an effective amount of a drug having an immunosuppressive effect. In a

5

10

fifteen

twenty

25

30

35

40

Four. Five

embodiment, the peptide has the amino acid sequence SEQ ID NO: 1, which comprises a phosphoserine at position 10 and a drug that has an immunosuppressive effect.

The peptide consisting of the amino acid sequence SEQ ID NO: 1, in which the serine in position 10 is phosphorylated, corresponds to Compound III shown below:

image6

As used herein, "a drug that has an immunosuppressive effect or an immunomodulatory effect" is a drug that inhibits or prevents the activity of a subject's immune system (for example, a mammal such as a human being). For example, drugs "that have an immunosuppressive effect" include: corticosteroids such as methyl prednisolone; cyclophosphamides; azathioprines; hydroxychloroquines; antimalarials; mycophenolate mofetil; methotrexate; biological agents, for example, infliximab, etanercept, golimumab, adalimumab, certolizumab; and combinations of the above. This list is not intended to be limiting of the invention. For example, the expert can easily determine other known drugs that have an immunosuppressive or immunomodulatory effect, their effective amounts and their routes of administration.

In certain embodiments, the description provides a therapeutic composition comprising an effective amount of a peptide having the amino acid sequence indicated in at least one of SEQ ID NO: 4, 5, or a combination thereof (i.e., that It comprises peptides of SEQ ID NO: 5 and SEQ ID NO: 4) and an effective amount of a drug having an immunosuppressive or immunomodulatory effect. In further embodiments, the composition may further comprise a peptide having the amino acid sequence indicated in SEQ ID NO: 1, which comprises a phosphoserine at position 10.

In another aspect, the description provides a composition as described herein for use in a method for treating an autoimmune disease comprising the step of administering to a patient in need of an effective amount of a pharmaceutical composition as described in this document, where the composition is sufficient to effect said treatment.

In certain embodiments, autoimmune disease is chosen from: autoimmune pathologies of the family of connective tissue diseases (non-organ-specific systemic diseases), for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, mixed connective tissue disease, syndrome of Sjogren or chronic juvenile arthritis; and / or organ-specific autoimmune pathologies, for example, multiple sclerosis, insulin dependent diabetes, Crohn's disease or bulosic diseases. In a preferred embodiment, the autoimmune disease is SLE.

The description also provides a drug comprising a peptide as described herein and / or a combination as described herein, for use as a drug, in particular for the treatment of autoimmune diseases.

In certain embodiments, autoimmune disease is chosen from: autoimmune pathologies of the family of connective tissue diseases (non-organ-specific systemic diseases), for example, systemic lupus erythematosus (SLE), rheumatoid arthritis, mixed connective tissue disease, syndrome of Sjogren or chronic juvenile arthritis; and / or organ-specific autoimmune pathologies, for example, multiple sclerosis, insulin dependent diabetes, Crohn's disease or bulosic diseases. In a preferred embodiment, the autoimmune disease is SLE.

The description also provides pharmaceutical compositions comprising at least one peptide as described herein, or a combination product as described above, which also includes a pharmaceutically acceptable carrier.

Peptides (also referred to herein as "active compounds"), as described herein, may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise peptide and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable vehicle" is intended to include each and every solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delayed absorption agents and the like, compatible with administration. Pharmaceutical The use of such media and agents for pharmaceutically active substances is well known in the art. Except when any means

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

or conventional agent is incompatible with the active compound, its use in the compositions is contemplated. Complementary active compound compositions can also be incorporated into the compositions.

The description provides methods for preparing pharmaceutical compositions. Such methods comprise the formulation of a pharmaceutically acceptable vehicle with a peptide as described herein. Such compositions may also include additional active agents as described above. Thus, the invention also includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with a peptide as described herein, and one or more additional active compounds.

A pharmaceutical composition of the invention is formulated to be compatible with the desired route of administration. Examples of routes of administration include parenteral administration, for example, intravenous, intradermal, subcutaneous, oral, nasal (eg, inhalation), transdermal (topical), transmucosal and rectal. The solutions or suspensions used for parenteral, intradermal or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparation can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline solution, bacteriostatic water, Cremophor EL (BASF, Parsippany, N. J.) or phosphate buffered saline solution (PBS). In all cases, the composition must be sterile and must be fluid to such an extent that it can be easily administered through a syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the necessary particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, ascorbic acid and the like. In many cases, it will be preferable to include in the composition isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Prolonged absorption of the injectable compositions can be produced by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (for example, a polypeptide or antibody) in the necessary amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filtration sterilization. In general, the dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium, and then incorporating the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, which produces a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. .

Oral compositions generally include an inert diluent or an edible vehicle. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a liquid vehicle for use as a mouthwash, where the compound in the liquid vehicle is applied orally and used to rinse and spit or swallow.

For administration by inhalation, the compounds are released in the form of an aerosol spray from a pressurized dispenser or container containing a suitable propellant, for example, a gas such as carbon dioxide or a nebulizer.

Systemic administration can also be performed by a transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be infiltrated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be performed through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, ointments, gels or creams as is generally known in the art.

The compounds can also be prepared in the form of suppositories (for example, with suppository bases

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

conventional such as cocoa butter and other glycerides) or retention enemas for rectal administration.

In one embodiment, the active compounds are prepared with vehicles that will protect the compound against rapid removal from the body, such as a controlled release formulation, including implants and microencapsulated release systems. Biodegradable and biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. The methods for preparing said formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes having monoclonal antibodies incorporated therein or on them) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Patent No. 2 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in the form of dosage units to facilitate administration and achieve uniformity in dosage. The dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification for the dosage unit forms of the invention is dictated by and is directly dependent on the unique characteristics of the active compound and the particular therapeutic effect that is desired to be achieved, and on the intrinsic limitations in the composition technique of said compound. active for the treatment of individuals.

In certain embodiments of the methods provided herein, the method includes the step of administering a dosage of about 100 ng to about 5 mg of a therapeutic or pharmaceutical composition as described herein. In certain embodiments, for example, in humans, the pharmaceutical composition as described herein may contain mannitol as a vehicle, and the composition is administered from 10 gg to 500 gg, preferably 200 gg, in a single administration.

In certain additional aspects, the dosage regimen can be reproduced 1 to 3 times / week, from every week to once every four weeks, for a period as long as necessary, with therapeutic windows and, therefore, for several years . In a preferred embodiment, the dosage regimen is 12 weeks of treatment, but can be repeated twice a year for several years. An example of administration is: an injection of 200 gg of peptide, every 4 weeks, for 12 weeks (that is, 3 injections separated from each other for 4 weeks).

The treatment can be prolonged by administration every 6 months.

Preferred pharmaceutically acceptable carriers may comprise, for example, xanthan gum, locust bean gum, galactose, other saccharides, oligosaccharides and / or polysaccharides, starch, starch fragments, dextrins, British gum and mixtures thereof. Advantageously, the pharmaceutically acceptable vehicle is of natural origin. The pharmaceutically acceptable vehicle may be, or may also comprise, an inert saccharide diluent selected from a monosaccharide or disaccharide. A preferred saccharide is mannitol.

Advantageously, the invention relates to a pharmaceutical composition as defined above, which is in the form of a dragee, tablet, gelatin, capsule, drop, pill, liposome or nanoparticles, or in the form of a solution. An advantageous solution is a solution comprising from 5 to 15%, in particular about 10% mannitol. The solution must be isoosmolar.

The invention also relates to a drug comprising a combination product as defined above, for simultaneous, separate or sequential use.

Examples

Statistics

Statistical tests were performed using GraphPad Prism version 5.0. The two-way ANOVA assay was used to analyze the statistical significance of proteinuria differences between groups of control and peptide treated mice. The survival of female MRL / lpr control mice treated with a P140 analog was analyzed by the Kaplan-Meier method and the significance of the differences was determined by the log-rank test. For the other variables, the statistical significance was evaluated using the Student's t-test. P values less than 0.05 were considered significant.

5

10

fifteen

twenty

25

Example 1: Chemical synthesis of peptides.

Peptide P140 and P140 (MO) were synthesized using Fmoc solid phase classical chemistry (N- [9-fluorenyljmethoxycarbonyl) and purified by reverse phase high performance liquid chromatography (HPLC, Neimark and Briand, 1993, Monneaux et al. ., 2003, Eur. J. Immunol., 33, 287-296, Page et al., 2009, PloS ONE 4, e5273). Its homogeneity was verified by analytical HPLC and its identity was evaluated by LC / MS in a Finnigan LCQ Advantage Max system (Thermo Fischer Scientific). After completion of the reaction, the peptides were purified by HPLC.

To introduce phosphorylation into the serine moiety equivalent to moiety 140 of SEQ ID NO: 3, a serine derivative of the Fmoc-Ser (PO (Obz) OH) -OH type was used. The coupling time is increased to 30 minutes and a second coupling is performed systematically. After cleavage in acidic medium, each peptide is precipitated by cold ether, solubilized in a solution of water and acetonitrile and finally lyophilized. The peptides are then purified by RP-HPLC, and their integrity and purity are analyzed by analytical HPLC and by mass spectrometry (Malditof).

The oxidation is introduced as mentioned above.

Example 2: stability of the peptides.

The stability of the peptide SEQ ID NO: 2 in which the serine in position 10 is phosphorylated and the methionine in position 4 is oxidized (P140 (MO)) and the stability of the peptide SEQ ID NO: 1 in which the serine at position 10 this phosphorylated (P140) was measured at 37 ° C, in a 10% (v / v) mannitol solution. For each peptide, 3 concentrations have been tested: 200, 100 and 50 pg / ml.

At the indicated time point, the integrity of peptides P140 and P140 (MO) in saline solution was measured by high performance liquid chromatography from the peak area corresponding to the intact peptide.

The results are shown in figure 1.

The following tables 1 and 2 summarize the results:

Table 1

 P140 (MO) P140

 Stability (%)

 Days 200 pg / ml 100 pg / ml 50 pg / ml 200 pg / ml 100 pg / ml 50 pg / ml

 0

 100 100 100 100 100 100

 twenty

 100 99.1 100 98.7 97.5 95.5

 40

 100 99.5 100 98.5 96.2 93.2

 60

 - - - 97.9 95.5 91.5

 80

 - - - 97.6 94.5 90.3

 100

 100

 99.1 99.4 97.4 93.4 89.6

Table 2

 P140 (MO) P140

 Stability (%)

 Days 200 pg / ml 100 pg / ml 50 pg / ml 200 pg / ml 100 pg / ml 50 pg / ml

 Linear equation

 y = 100 y = - 0.0064x + 100.11 y = - 0.0064x + 99.677 y = - 0.0238x + 99.535 y = - 0.0612x + 99.25 y = - 0.099x + 98.299

 Correlation coefficient

 N / A R2 = 0.8571 R2 = 0.4157 R2 = 0.8854 R2 = 0.9538 R2 = 0.9065

 95% stability (predicted)

 00 2 years + 2 months 2 years 6 months 2 months 1 month

5

10

fifteen

twenty

25

30

35

40

Four. Five

Stability is measured using the HPLC peak surface.

The stability of P140 M (O) remains unchanged (100%, 99.1% and 99.4%) for 100 days at 37 ° C, for each of the concentrations tested (50 to 200 pg / ml).

The stability of P140 is reduced over time and reduced after 100 days at 37 ° C (97.4%, 93.4% and 89.6%) for each of the concentrations tested (from 50 to 200 pg / ml).

These data demonstrate that the oxidation of methionine in peptide P140 increases the stability of the peptide. P140 (MO) is stable at all concentrations tested for more than 100 days.

Example 2: therapeutic effect of peptides in MRL / lpr mice.

The MRL / lpr mouse strain is a mouse sub-strain that is genetically predisposed to the development of a systemic lupus erythematosus-like syndrome that, as noted, is clinically similar to human disease. It has been determined that this mouse strain carries a mutation in the fas gene. In addition, the MRL / lpr is a useful model to study behavioral and cognitive deficits found in autoimmune diseases and the efficacy of immunosuppressive agents [Monneaux et al., 2003, Eur. J. Immunol. 33, 287-296].

2.1- Survival analysis

Five-week-old female MRL / lpr mice received P140 or P140 peptide (MO) intravenously as described (Monneaux et al., 2003, Eur. J. Immunol., 33, 287-296). All experimental protocols were carried out with the approval of the local Institutional Committee for Animal Care and Use (CREMEAS). As a control, mice are injected with NaCl.

20 mice were used for each peptide or NaCl.

The results are shown in figure 2.

A Log-rank test (Mantel-Cox) has been applied and the results are as follows: NaCl vs P140 p = 0.0686, NaCl vs P140 (MO) p = 0.0026, P140 vs P140 M (O) p = 0.2366.

The median survival of the mice is: NaCl = 25 weeks, P140 = 29 weeks and P140 (MO)> 40 weeks. These results demonstrate the efficacy of peptide P140 (MO) in vivo in the treatment of lupus, in mice.

2.2- Proteinuria analysis

The proteinuria of the previous mice in freshly collected urine was measured using Albustix (Bayer Diagnostics) and was estimated semi-quantitatively according to a 0-4 scale recommended by the manufacturer (absence of proteinuria = 0; traces = 1; 1 + = 2 ; 2 + = 3; 3 + = 4; 4 + = 5).

The results are shown in figure 3.

In this figure, it is observed that proteinuria is less important and appears late in mice treated with P140 M (O) compared to untreated mice.

2.3- Cellularity analysis

In MRL / lpr mice 100 pg / 100 pl of P140 or P140 (MO) were injected and the cellularity (peripheral blood) was studied 5 days after this single injection. The count includes all leukocytes. Due to the low number of mice tested, a non-parametric Mann-Whitney statistical test has been performed).

The results are shown in figure 4.

Thus, in an acute murine model of lupus, the peptide of SEQ ID NO: 4 was able to reduce peripheral hypercellularity and delay the biological and clinical signs of the disease with an efficacy at least similar to that of P140, or better.

Example 3: affinity of the peptides for the HSC70 protein.

A BIAcore 3000 (Biacore AB) system was used to evaluate the binding of P140 peptides to the HSC70 protein (Page et al., 2009 and 2011). The CM5 detection microplate, the P20 surfactant, the amine coupling kit containing N-hydroxysuccinimide (NHS) and N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC), 2- (2-pyridinyldithio) ethanolamine (PDEA) and ethanolamine They came from Biacore AB. The biosensor assays were performed with HBS-EP buffer as a processing buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant, pH 7.4). The compounds were diluted in the processing buffer. The surface of the sensor microplate is regenerated after each experiment by injecting 10 pl of 10 mM HCl. Bovine recombinant HSC70 (Stressgen) was immobilized in flow cells of a CM5 sensor microplate through its thiol groups using 35 pl of PDEA in

50 mM borate buffer, pH 8.3 in the matrix activated by NHS / EDC. Then, 35 pi of HSC70 (100 pg / ml in formate buffer, pH 4.3) was injected until a response of 13,000 response units (UR) corresponding to 13 ng / mm2 of HSC70 was immobilized. 20 pi of a 50 mM cysteine solution / 1 M NaCi were used to saturate the unoccupied sites in the microplate. The direct union measurement of peptides P140 to HSC70 was carried out at 25 ° C with a constant flow rate of 20 pl / min. Peptide P140 and its analogs were injected into the flow at different concentrations for 3 minutes, followed by a 3 minute dissociation phase. Kinetic parameters were calculated using BIAeval 3.1 software on a personal computer. The analysis was performed using the simple 1: 1 Langmuir union model. Specific binding profiles were obtained after subtracting the response signal from the empty control channel and from the injection with control buffer. The adjustment to each model was judged by the 10 x2 value and the randomness of the distribution of remains compared to the theoretical model.

The results are shown in tables 3 and 4, and in figures 5 and 6.

These tables demonstrate that the affinity for HSC70 is not statistically different between peptides P140 and P140 M (O).

In this way, these two peptides bind with the same efficacy to HSC70.

fifteen

Table 3: P140 in HSC70

 Chi2

 3.17

 kobs (1 / s)

   CO or 1 LU CM 00 co ~ CO or 1 LU C \ l io sr CO O i LU CO CD LO 8.74E-03 0.0144

 Req (RU)

   15.3 25.8 39.5 53.6 65.2

 KD (M)

   to 1 LU CD CD CO 1 LU CD CD CO 1 LU CD CO "CO 1 LU CD CD CO 1 LU CD CD

 KA (1 / M)

   1.44E + 05 1.44E + 05 1.44E + 05 1.44E + 05 1.44E + 05

 Cone, analyte

   1.56 or 3.12 or 6.25 or 12.5 or 25 u

 Rl (RU)

   12.1 CD or 'CSI 33.8 62.5 118

 Rmax (RU)

 CO CO 00

 kd (1 / s)

   CO or 1 LU CM T— CO ~ CO O i LU CM T— co ~ CO or 1 LU CM t— CO ~ CO or 1 LU CM T— co ~ CO or 1 LU CM t— co ~

 ka (1 / Ms)

 450

 Peptide - concentration

   3. CO IO 1 or TT £ CM with "i or £ 3. LO CM CD i O £ 3. LO cm" 1 or sj- £ 3. IO CM i O £

Table 4: P140 (MO) in HSC70

 Peptide - concentration

 ka (1 / Ms) kd (1 / s) Rmax (RU) RI (RU) Cone, analyte KA (1 / M) KD (M) Req (RU) kobs (1 / s) Chi2

 1.15E + 3 39 1.18

 P140 (MO) -1.56 pM

   2.20E-3 14 1.56 or 5.24E + 5 1.91 E-6 17.6 4.00E-03

 P140 (MO) -3.12 pM

   2.20E-3 18.7 3.12 or 5.24E + 5 1.91E-6 24.2 5.80E-03

 P140 (MO) -6.25 pM

   2.20E-3 25.9 6.25 or 5.24E + 5 1.91E-6 29.9 9.40E-03

 P140 (MO) -12.5 pM

   2.20E-3 36.9 12.5 or 5.24E + 5 1.91E-6 33.9 0.0166

 P140 (MO) -25 pM

   2.20E-3 53.4 25 or 5.24E + 5 1.91E-6 36.3 0.031

5

10

fifteen

twenty

25

30

35

40

<110> NATIONAL CENTER OF THE RECHERCHE SCIENTIFIQUE IMMUPHARMA France SA MULLER, Sylviane BRIAND, Jean-Paul ZIMMER, Robert

<120> MODIFIED PEPTIDES AND ITS USE TO TREAT AUTOIMMUNITY DISEASES <130> IWB 11 CJ CNR P140 <160> 6

<170> PatentIn version 3.5

<210> 1 <211> 21 <212> PRT

<213> Artificial sequence <220>

<223> derived from U1 snRNP 70 kDa <400> 1

Arg lie His Met Val Tyr Ser Lys Arg Ser Gly Lys Pro Arg Gly Tyr 15 10 15

Phe lie Glu Tyr 20 wing

<210> 2 <211> 20 <212> PRT

<213> Artificial sequence <220>

<223> derived from U1 snRNP 70 kDa <400> 2

Lie His Met Val Tyr Ser Lys Arg Ser Gly Lys Pro Arg Gly Tyr Ala 15 10 15

Phe lie Glu Tyr 20

<210> 3 <211> 437 <212> PRT <213> Homo sapiens

<400> 3

Met Thr Gin Phe Leu Pro Pro Asn Leu Leu Ala Leu Phe Ala Pro Arg 15 10 15

Asp Pro lie Pro Tyr Leu Pro Pro Leu Glu Lys Leu Pro His Glu Lys 20 25 30

His His Asn Gin Pro Tyr Cys Gly lie Ala Pro Tyr lie Arg Glu Phe 35 40 45

Glu Asp Pro Arg Asp Ala Pro Pro Pro Thr Arg Ala Glu Thr Arg Glu 50 55 60

Glu Arg Met Glu Arg Lys Arg Arg Glu Lys lie Glu Arg Arg Gin Gin 65 70 75 80

Glu Val Glu Thr Glu Leu Lys Met Trp Asp Pro His Asn Asp Pro Asn 85 90 95

Wing Gin Gly Asp Wing Phe Lys Thr Leu Phe Val Wing Arg Val Asn Tyr 100 105 110

Asp Thr Thr Glu Ser Lys Leu Arg Arg Glu Phe Glu Val Tyr Gly Pro 115 120 125

He Lys Arg lie His Met Val Tyr Ser Lys Arg Ser Gly Lys Pro Arg 130 135 140

Gly Tyr Ala Phe lie Glu Tyr Glu His Glu Arg Asp Met His Ser Ala 145 150 155 160

Tyr Lys His Wing Asp Gly Lys Lys lie Asp Gly Arg Arg Val Leu Val 165 170 175

Asp Val Glu Arg Gly Arg Thr Val Lys Gly Trp Arg Pro Arg Arg Leu 180 185 190

Gly Gly Gly Leu Gly Gly Thr Arg Arg Gly Gly Ala Asp Val Asn lie 195 200 205

Arg His Ser Gly Arg Asp Asp Thr Ser Arg Tyr Asp Glu Arg Pro Gly 210 215 220

Pro Ser Pro Leu Pro His Arg Asp Arg Asp Arg Asp Arg Glu Arg Glu 225 230 235 240

Arg Arg Glu Arg Ser Arg Glu Arg Asp Lys Glu Arg Glu Arg Arg Arg 245 250 255

Be Arg Be Arg Asp Arg Arg Arg Arg Be Arg Be Arg Asp Lys 260 265 270

Glu

image7

<210> 4 <211> 21

5 <212> PRT

<213> Artificial sequence

<220>

<223> derived from U1 snRNP 70 kDa

10

<220>

<221> MOD_RES <222> (4) .. (4)

<223> OXIDATION 15

<220>

<221> MOD_RES <222> (10) .. (10)

<223> FOSFORILZATION

<400> 4

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

Arg lie His Met Val Tyr Ser Lys Arg Ser Gly Lys Pro Arg Gly Tyr 15 10 15

Phe lie Glu Tyr wing

twenty

<210> 5 <211> 20 <212> PRT

<213> Artificial sequence <220>

<223> derived from U1 snRNP 70 kDa <220>

<221> MOD_RES <222> (3) .. (3)

<223> OXIDATION

<220>

<221> MOD_RES <222> (9) .. (9)

<223> FOSFORILZATION

<400> 5

Lie His Met Val Tyr Ser Lys Arg Ser Gly Lys Pro Arg Gly Tyr Ala 15 10 15

Phe lie Glu Tyr 20

<210> 6 <211> 66 <212> DNA

<213> Artificial sequence <220>

<223> derived from U1 snRNP 70 kDa <220>

<221> misc_feature <222> (3) .. (3)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (15) .. (15)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (21) .. (21)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (27) .. (27)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (30) .. (30)

5

10

fifteen

twenty

25

30

<223> n is a, c, g or t <220>

<221> misc_feature <222> (33) .. (33)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (39) .. (39)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (42) .. (42)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (45) .. (45)

<223> n is a, c, g or t

<220>

<221> misc_feature <222> (51) .. (51)

<223> n is a, c, g or t

<400> 6

mgnathcaya tggtntayws naarmgnwsn ggnaarccnm gnggntaygc nttyathgar 60

taytrr 66

Claims (6)

1. A peptide, or salt thereof, that comprises or consists of the amino acid sequence of SEQ ID NO: 2 IHMVYSKRSGKPRGYAFIEY [SEQ ID NO: 2], which comprises a phosphoserine at position 9 and an oxidized methionine at position 3. A peptide, or salt thereof, which comprises or consists of the amino acid sequence of SEQ ID NO: 1 RIHMVYSKRSGKPRGYAFIEY [SEQ ID NO: 1], comprising a phosphoserine at position 10 and an oxidized methionine at position 4. 3. A composition comprising a peptide according to claim 1 or 2, or both, and a drug having an immunosuppressive effect or an immunomodulatory effect. A pharmaceutical composition comprising a peptide according to claims 1, 2, 3 or a combination thereof, which also comprises a pharmaceutically acceptable vehicle. 5. The pharmaceutical composition according to claim 4, which is in the form of a dragee, tablet, gelatin, capsule, drop, pill, liposome, nanoparticles or in the form of a solution. 6. A pharmaceutical composition according to claim 4 or 5 for use in the treatment of an autoimmune disease. 7. The pharmaceutical composition for use according to claim 6, wherein said autoimmune disease is selected from the group consisting of autoimmune pathologies of the family of connective tissue diseases (non-organ-specific systemic diseases), preferably systemic lupus erythematosus (SLE), rheumatoid arthritis, mixed connective tissue disease, Sjogren's syndrome, juvenile arthritis 20 chronicle; and organ-specific autoimmune pathologies, preferably multiple sclerosis, insulin dependent diabetes, Crohn's disease and bulosic diseases. 8. The pharmaceutical composition for use according to claim 6 or 7, wherein said autoimmune disease is systemic lupus erythematosus (SLE).